Pretreatment of animals with cyclophosphamide (CY) can augment cell-mediate immunity to tumor-associated antigens. This immunopotentiating effect is probably caused by selective toxicity for T lymphocyte-mediated suppressor activity. We have shown that pretreatment of patients with advanced cancer with CY, either 1000 or 300 mg/M2, significantly augmented the development of delayed-type hypersensitivity (DTH) to the primary antigen, keyhole limpet hemocyanin (KLH). Furthermore, we observed that administration of CY 3 days prior to injection of an autologous tumor vaccine resulted in the acquisition of DTH to the autologous tumor cells in a group of patients with metastatic melanoma who did not become immunized without CY. To date, treatment with CY + vaccine has resulted in clinically important regression of metastases in 4/33 patients. Studies with peripheral blood lymphocytes (PBL) from these patients indicate that CY inhibits the generation of T suppressor cells, possibly by toxicity for a suppressor-inducer cell with the phenotype, CD4+,2H4+. We now propose to continue and extend these studies: Specific aim 1) is to identify and characterize melanoma antigen-responsive suppressor T cells. We will exploit a recently developed method to test the effect of putative suppressors on in vitro responses to melanoma cells and an established technique utilizing suppressors elicited by melanoma cells but tested in a mitogen-activated indicator system. Specific aim 2) is to determine whether heterogeneity of expression of melanoma-associated antigens is an important limitation to the effectiveness of active immunotherapy and whether it can circumvented by therapeutic modification. Specific aim 3) is test the hypothesis that the response to tumor antigens can be augmented by immunizing with trinitrophenol-conjugated tumor cells in the absence of suppression, thereby inducing a population of amplified helper cells or possibly contrasuppressor cells. Finally, Specific aim 4) is an attempt to take advantage of the concept that tolerance to self-antigens, and perhaps tumor antigens as well, is HLA- restricted and can be reversed by presenting those antigens in the context of foreign HLA products, i.e., on allogeneic cells. We expect these studies to lead to more effective immunotherapy of human melanoma.